Efforts to elucidate the particular regions and residues of Escherichia coli heat-labile enterotoxin involved in toxic and enzymatic activity have continued. Based on the recently published crystal structure that predicts the position of an NAD-binding site in the A subunit, we have subjected a number of residues to site directed mutagenic alteration. Analyses of the mutant analogs has revealed that in most cases alterations which abrogate ADP-ribosyltransferase activity also impart conformational changes that are detectable by tryptic fragmentation patterns. However, conservative substitution at tryptophan 170 by tyrosine yields a molecule with reduced activity and apparent wild-type conformation. This mutation, in conjunction with other previously described substitutions may be a suitable target for the generation of, multiply substituted mutant proteins that are devoid of toxic/enzymatic activity and that retain native conformation. We are continuing to investigate various amino acid substitutions at various target in the hope of generating mutants with the appropriate phenotypic characteristics (i.e. no enzymatic activity and no gross conformational alterations). Additional studies have been conducted to evaluate the effect of post- translational modifications on the enzymatic activity of the A subunit of LT. These studies have revealed that limited proteolytic cleavage is not necessary for the expression of enzymatic activity and that multiple auto-or self ADP-ribosylation does not detectably change the specific enzymatic activity of the toxin. These findings contrast the results of studies conducted by other groups using cholera toxin.